The genes encoding U1 small nuclear RNA have been isolated from the chicken (Roop et al., 1981) and human genomes (Manser and Gesteland, 1981; Lund and Dahlberg, personal communication; our unpublished work). The presence of a cap structure on the 5' end of U1 snRNA strongly suggests that it is an RNA polymerase II product, and biosynthesis of U1 is accordingly sensitive to alpha-amanitin (Jensen et al., 1979) and the drug DRB (Tamm et al., 1980); however, all attempts to transcribe these U1 snRNA genes in vitro have failed so far. Here we propose a strategy for analyzing the transcription of cloned U1 genes by first determining what DNA sequences are necessary for transcription to occur in vivo, and then applying this information to guide subsequent studies in vitro. U1 genes which we have previously isolated will be cloned into the vector, pSV08 (Meyers and Tjian, 1980). Plasmid pBR322 sequences in pSVO8 enable it to replicate in E. coli, and a 311 base pair SV40 origin sequence enable it to replicate in COS-7 monkey cells (Gluzman, 1981) which constitutively express T-antigen from an integrated origin-defective copy of SV40. When introduced into the COS-7 host, pSV08 replicates without restraint (Proudfoot et al., 1981) and effectively converts a single copy U1 locus into a highly repetitive gene whose transcripts can be monitored above a background of U1 transcripts derived from the host's own multigene family. We will use this approach to (1) define the boundaries of the U1 transcription unit in vivo by constructing systematic deletions of the U1 gene, (2) determine the size of primary U1 transcript in vivo, and (3) identify transcriptional punctuation signals in the U1 gene by directed in vitro mutagenesis. In particular, the absence of an upstream TATA box in both chicken and human U1 genes (op. cit.) bears investigation, as does the nature of the termination and/or processing signal for this short nonpolyadenylated RNA polymerase II transcript. We have at least 9 distinct U1 genes to which this analysis can be applied, as well as 10 human U2 genes. With the sophistication we hope to gain from these in vivo studies, we will then attempt to modify and manipulate the available RNA polymerase II in vitro transcription system (Weil et al., 1979; Manely et al., 1980; Weingartner and Keller, 1981) to characterize U1 transcription in greater detail.